TEM analysis and IFA of fusion-GEM complexes
For transmission electron microscopy (TEM) observation, the fusion-GEM complexes were centrifuged to obtain the precipitate. After fixation with 2% glutaraldehyde (no suspension) overnight at 4°C, the precipitate was sliced into ultrathin sections. Then, the samples were stained with uranium acetate and lead citrate, and their features were observed under a transmission electron microscope.
For the IFA, the precipitate from centrifuged fusion-GEM complexes was suspended and blocked with 5% nonfat milk. Then, the cells were stained with an anti-flavivirus E mAb (1:200) as the primary antibody and with FITC anti-mouse IgG (1:200) as the secondary antibody. The stained complexes were applied to slides and observed under a fluorescence microscope (Olympus Corp, Tokyo, Japan).